aberystwyth university benefits of condensed tannins in
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Aberystwyth University
Benefits of condensed tannins in forages fed to ruminantsMueller-Harvey, Irene; Bee, Guiseppe; Dohme-Meier, Frigga; Hoste, Hervé; Karonen, Maarit; Kölliker, Roland;Lüscher, Andreas; Niderkorn, Vincent; Pellikaan, Wilbert; Salminen, Juha-Pekka; Skot, Leif; Smith, Lydia;Thamsborg, Stig; Totterdell, Paul; Wilkinson, Ian; Williams, Andrew; Azuhnwi, Blasius; Baert, Nicolas; Brinkhaus,Anja Grosse; Copani, GiuseppePublished in:Crop Science
DOI:10.2135/cropsci2017.06.0369
Publication date:2019
Citation for published version (APA):Mueller-Harvey, I., Bee, G., Dohme-Meier, F., Hoste, H., Karonen, M., Kölliker, R., Lüscher, A., Niderkorn, V.,Pellikaan, W., Salminen, J-P., Skot, L., Smith, L., Thamsborg, S., Totterdell, P., Wilkinson, I., Williams, A.,Azuhnwi, B., Baert, N., Brinkhaus, A. G., ... Waghorn, G. (2019). Benefits of condensed tannins in forages fed toruminants: importance of structure, concentration and diet. Crop Science, 59, 1-25.https://doi.org/10.2135/cropsci2017.06.0369
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crop science, vol. 59, 2019 www.crops.org 1
REviEw & intERpREtation
Despite the widespread occurrence of condensed tannins (CTs) in the plant kingdom (Fig. 1), there are still large gaps in our
knowledge that continue to challenge plant breeding, animal sci-ence, and analytical chemistry; progress in all of these disciplines is needed in order understand the mechanisms that underpin their actions and to fully exploit their benefits.
Benefits of Condensed Tannins in Forage Legumes Fed to Ruminants: Importance of
Structure, Concentration, and Diet CompositionIrene Mueller-Harvey,* Giuseppe Bee, Frigga Dohme-Meier, Hervé Hoste, Maarit Karonen, Roland Kölliker, Andreas Lüscher, Vincent Niderkorn, Wilbert F. Pellikaan, Juha-Pekka Salminen, Leif Skøt, Lydia M.J. Smith, Stig M. Thamsborg, Paul Totterdell, Ian Wilkinson, Andrew R. Williams, Blasius N. Azuhnwi, Nicolas Baert, Anja Grosse Brinkhaus, Giuseppe Copani, Olivier Desrues, Chris Drake, Marica Engström, Christos Fryganas, Marion Girard, Nguyen T. Huyen, Katharina Kempf, Carsten Malisch, Marina Mora-Ortiz, Jessica Quijada,
Aina Ramsay, Honorata M. Ropiak, and Garry C. Waghorn
ABSTRACTCondensed tannins (CTs) account for up to 20% of the dry matter in forage legumes used as rumi-nant feeds. Beneficial animal responses to CTs have included improved growth, milk and wool production, fertility, and reduced methane emis-sions and ammonia volatilization from dung or urine. Most important is the ability of such forages to combat the effects of gastrointestinal parasitic nematodes. Inconsistent animal responses to CTs were initially attributed to concentration in the diet, but recent research has highlighted the importance of their molecular structures, as well as concen-tration, and also the composition of the diet con-taining the CTs. The importance of CT structural traits cannot be underestimated. Interdisciplinary research is the key to unraveling the relationships between CT traits and bioactivities and will enable future on-farm exploitation of these natural plant compounds. Research is also needed to provide plant breeders with guidelines and screening tools to optimize CT traits, in both the forage and the whole diet. In addition, improvements are needed in the competitiveness and agronomic traits of CT-containing legumes and our understanding of options for their inclusion in ruminant diets. Farm-ers need varieties that are competitive in mixed swards and have predictable bioactivities. This review covers recent results from multidisciplinary research on sainfoin (Onobrychis Mill. spp.) and provides an overview of current developments with several other tanniniferous forages. Tannin chemistry is now being linked with agronomy, plant breeding, animal nutrition, and parasitology. The past decade has yielded considerable prog-ress but also generated more questions—an envi-able consequence of new knowledge!
I. Mueller-Harvey, C. Drake, C. Fryganas, A. Ramsay, and H.M. Ropiak, School of Agriculture, Policy and Development, Univ. of Reading, PO Box 236, Reading RG6 6AT, UK; G. Bee, F. Dohme-Meier, A.G. Brinkhaus, and M. Girard, Agroscope-Posieux, CP 64, 1725 Posieux, Switzerland; H. Hoste, B.N. Azuhnwi, and J. Quijada, INRA, UMR 1225 IHAP, BP 87614, Toulouse 31076, France; M. Karonen, J.-P. Salminen, N. Baert, and M. Engström, Dep. of Chemistry, Univ. of Turku, 20014 Turku, Finland; R. Kölliker, Molecular Plant Breeding, ETH Zürich, 8092 Zürich, Switzerland; A. Lüscher and C. Malisch, Forage Production and Grassland Systems, Agroscope, 8046 Zürich, Switzerland; V. Niderkorn and G. Copani, INRA UMR1213 Herbivores, 63122 St-Genès-Champanelle, France; W.F. Pellikaan and N.T. Huyen, Wageningen Univ. and Research, Animal Nutrition Group, PO Box 338, 6700 AH Wageningen, the Netherlands; L. Skøt, IBERS, Aberystwyth SY23 3EB, UK; L.M.J. Smith and M. Mora-Ortiz, NIAB, Huntingdon Rd., Cambridge CB3 0LE, UK; S.M. Thamsborg, A.R. Williams, and O. Desrues, Dep. of Veterinary Disease Biology, Univ. of Copenhagen, Dyrlægevej 100, Frederiksberg C 1870, Denmark; P. Totterdell and I. Wilkinson, Cotswold Seeds, London Rd., Moreton-in-Marsh GL56 0JQ, UK; K. Kempf, Molecular Ecology, Agroscope, 8046 Zürich, Switzerland; G.C. Waghorn, Institute of Veterinary and Biomedical Sciences, Massey Univ., Private Bag 11222, Palmerston North 4474, New Zealand. Received 19 July 2017. Accepted 18 Oct. 2017. *Corresponding author ([email protected]). Assigned to Associate Editor Jose Dubeux.
Abbreviations: BSA, bovine serum albumin; CT, condensed tannin; mDP, mean degree of polymerization; MYB, myeloblastosis; NIRS, near-infrared reflectance spectroscopy; PC, procyanidin; PD, prodelphinidin; PEG, polyethylene glycol; RuBisCo, ribulose-1,5-bisphosphate carboxylase/oxygenase; SSR, simple sequence repeat.
Published in Crop Sci. 59:1–25 (2019). doi: 10.2135/cropsci2017.06.0369 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published online November 29, 2018
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This review focuses on progress achieved during the past decade and considers the following topics:
1. bioactive CTs in plants in general and forage legumes in particular;
2. intra- and interspecies variations in CT contents and composition, which will be termed CT traits from here on;
3. effects of agronomic, harvesting, and processing practices on CT efficacies;
4. effects on animal health, nutrition, product quality, and environmental emissions; and
5. characteristics useful for plant breeders and tools for selecting or engineering forages with novel CT traits.
This review also summarizes results from a multidisciplinary research consortium that focused on sainfoin (Fig. 2). In this project, agronomists and plant breeders assembled germplasm collections of sainfoin (Onobrychis Mill. spp.), which is a traditional forage legume in Europe, and identified molecular markers and strategies for weed control. Ruminant nutritionists studied various accessions for their in vitro fermentation characteristics and in vivo feeding trials, nitrogen balances, and the quality of meat and dairy products. Parasitologists explored the antiparasitic properties of a wide range of CT traits, and chemists developed tools for analyzing the CTs in sainfoin pellets, silages, and digesta.
The background to this work was the pioneering research in New Zealand that first studied fresh sainfoin (Onobrychis viciifolia Scop.) and Lotus L. spp. in relation to legume bloat and nutritive value (Reid et al., 1974). Sainfoin research included measurements of the protein-binding characteristics of CTs (Fig. 1) ( Jones and Mangan, 1977) and aspects of nitrogen digestion in sheep (Egan and Ulyatt, 1980), but problems with sainfoin persistence in swards diverted attention to Lotus species. Initial research by Barry and colleagues focused on big trefoil (Lotus pedunculatus Cav.),
examining effects of CT concentration (Barry et al., 1986), whereas Waghorn et al. (1987) demonstrated the beneficial effects of CTs in birdsfoot trefoil (Lotus corniculatus L. var. corniculatus) on absorption of essential amino acids from the intestine. Other forages were also evaluated in New Zealand, including sulla (Hedysarum coronarium L.; Stienezen et al., 1996) and dock (Rumex obtusifolius L.; Waghorn and Jones, 1989), but the importance of CT composition, in addition to concentration, was demonstrated more recently (Waghorn et al., 1997). Subsequent research included CT effects on livestock parasites and greenhouse gas emissions, but financial support decreased because of a lack of competitiveness of tanniniferous forages, especially in fertile soils. It also became apparent that detailed chemical characterization was required to elucidate mechanisms of action, because the two Lotus species differed in their biological effects and tannin types. The question was: were the CTs responsible for these different biological effects?
This review seeks to provoke discussion on how to progress this area of research, how to optimize the
Fig. 1. Example of a condensed tannin (CT) molecule that consists of four flavan-3-ol subunits (= monomeric building blocks). Procyanidins are composed of catechin or epicatechin and prodelphinidins of gallocatechin or epigallocatechin subunits (for further details, see Zeller, 2019).
Fig. 2. The LegumePlus project—an interdisciplinary European Union-funded research and training network on sainfoin (http://legumeplus.eu).
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a few CT-containing feeds with ruminant animals (e.g., sheep, goats, and cattle; Mueller-Harvey, 2006; Waghorn, 2008). The nutritional benefits include improved growth, milk yields, fertility, and tolerance to some intestinal parasites and arise from protection of dietary protein from excessive fermentation in the rumen. Other benefits include bloat prevention, which is associated with tannins reducing the stability of a foam that traps ruminal fermentation gases, and antiparasitic effects against ruminant and nonruminant gastrointestinal parasites (Waghorn, 2008; Kingston-Smith et al., 2010; Terrill et al., 2012; Wang et al., 2012; Hoste et al., 2015, 2016; MacAdam and Villalba, 2015).
Although plants synthesize many different tannin types, this review will focus on CTs, as these are of particular interest in forage legumes and several other pasture plants. Hydrolyzable tannins are not considered here, although evidence is emerging that some may exert similarly useful bioactive effects (Baert et al., 2016; Engström et al., 2016; Bee et al., 2017).
A major impetus for research and utilization of CT forage legumes by livestock producers has been the drive to reduce bloat, to improve farm profitability, to control parasites, and to reduce greenhouse gas and ammonia emissions (Kingston-Smith et al., 2010; Wang et al., 2012; McCaslin et al., 2014; Hoste et al., 2015). Nutritional responses to CTs have been variable, and this has led to contradictory reports about their benefits (Min et al., 2003; Mueller-Harvey, 2006; Waghorn, 2008). This is not surprising given the complexity of plant CTs, their impact when forages are fed as a sole diet or as a dietary component, and their interactions with feed components, host tissues, and the microbiome, plus the effects stemming from the animal’s nutrient requirements and parasitism. A concerted multidisciplinary research approach is required to harness the full potential of CTs for livestock production (Mueller-Harvey, 2006; Waghorn, 2008). However, obtaining funding for such a wide-ranging set of topics has been challenging and reflects common barriers to interdisciplinary research, such as narrowly focused funding goals and short funding timelines. Progress in the study of complex plant–livestock systems is inherently slow, and CTs also pose interesting analytical and experimental challenges. Taken together, these facts account for the relatively slow progress in identifying the relationships between CT traits and bioactivities.
CHALLENGES AND OPPORTUNITIES POSED BY PLANTS WITH DIFFERENT CONDENSED TANNIN TYPESResearch on plant CTs has come a long way since the early literature described these compounds of mysterious composition and function as “accidents of [plant] metabolism” or metabolic “waste products” that served to
bioactivity of CT forages, and how to develop on-farm applications. It will also consider whether farmers might require plants with different CT traits for either antiparasitic or nutritional purposes. The reader is also referred to the accompanying article by Zeller (2019) for a detailed description of CT structures and the review by Tedeschi et al. (2014) for a mechanistic model describing the overall interactions between CTs and ruminants.
ROLES OF TANNINS IN PLANTS AND CHALLENGES TO HARNESSING THEIR BENEFITS FOR LIVESTOCK PRODUCTIONThe reader of the tannin literature is repeatedly reminded that tannins are “secondary” plant metabolites and provide a defense against herbivory (Barbehenn and Constabel, 2011; Agrawal et al., 2012; Lattanzio et al., 2012). However, herbivores comprise a range of species, from insects to ruminants, and have distinctly different gut systems. Although tannins can account for antiherbivory effects in insects (Salminen and Karonen, 2011), ruminant behavior suggests that their herbivory effect is marginal because forages containing CTs are consumed, and selection is often in preference to grasses (Waghorn, 2008). Within plants, leaves are selected in preference to stems despite higher CT concentrations, even when CTs accounted for ~20% of the dry matter in erect canary clover [Dorycnium rectum (L.) Ser.] leaf (Waghorn and Molan, 2001). Current thinking suggests that these secondary metabolites provide plants with a plasticity that can support their development and interaction with the environment (Bidel et al., 2010; Neilson et al., 2013; Mouradov and Spangenberg, 2014). This concept, that CTs may have multiple and interrelated functions, is now gaining traction. For example, Chen et al. (2014) showed that the temperatures experienced by the mother plant elicit CT pathways that can pass information to the next generation. In addition, the flavan-3-ol monomers, which are precursors of CTs, appear to play a vital role in protecting chromosomes during periods of high cell activity, but not during dormancy or drought stress (Feucht et al., 2013). It is important to appreciate that a multitude of different CT compounds exist (Khanbabaee and van Ree, 2001; Hümmer and Schreier, 2008; Salminen and Karonen, 2011; Zeller, 2019), and that their synthesis in the plant kingdom has not converged on a single structure. These findings suggest that the function of CTs deserves a closer look in plant and crop science.
From an animal’s perspective, when dietary CT concentrations are too high, or protein concentrations are too low, as in tropical environments where grasses may have little nitrogen and tree leaves may have high CT concentrations, CTs can be antinutritional (Cooper et al., 1988). The benefits of CTs have been demonstrated in only
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support the primary plant metabolism (Haslam, 1981). It is now well established that CT synthesis is under genetic control (Szczyglowski and Stougaard, 2008; Scioneaux et al., 2011; Cheynier et al., 2013; Escaray et al., 2014) and that expression depends on the plant species and plant parts (Larkin et al., 1997; Gebrehiwot et al., 2002; Abeynayake et al., 2012; Ferreyra et al., 2012; Hancock et al., 2012; Verdier et al., 2012; Cheynier et al., 2013; Harding et al., 2013; Mouradov and Spangenberg, 2014; Pérez-Díaz et al., 2014; Zhou et al., 2015; Zhu et al., 2015; Chezem and Clay, 2016).
Chemotaxonomic surveys on the distribution of CTs in plants in general and of forage legumes in particular have found that CT compositions tend to follow distinct biosynthetic patterns in terms of their flavan-3-ol subunit composition and polymer sizes, which are described in terms of mean degree of polymerization (mDP). Plants with procyanidin (PC)-type CTs are much more widespread than plants with prodelphinidin (PD)-type CTs (Fig. 1), but many more plant species contain PC/PD mixtures (Porter, 1988; Mueller-Harvey, 2006; Sivakumaran et al., 2006; Mechineni et al., 2014; Laaksonen et al., 2015; Quijada et al., 2015; Hoste et al., 2016; Ropiak et al., 2016b). Most plant CTs have cis-flavan-3-ol subunits, especially as extension units (Fig. 1), whereas CTs with predominantly trans-flavan-3-ol subunits in extension units are relatively rare (Porter, 1988; Hernes and Hedges, 2004; Klongsiriwet et al., 2013). We have also observed other trends in the composition of CTs in forage legumes, which are illustrated with examples from a few plants that are “specialists” in producing particular CT types:
• CTs that comprise PCs tend to be mixtures of oligomers and smaller polymers (e.g., cocoa [Theo-broma cacao L.] bean CTs with mDP values of 2–5).
• CTs that comprise PDs are usually mixtures of larger polymers (e.g., sericea lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don] CTs with mDP values of 10–30).
However, exceptions exist, as lime tree flowers (Tilia L. spp.) and some varieties of cider apples (Malus domestica Borkh.) have PCs with higher mDP values of 8 and ~100, respectively (Guyot et al., 2001a; Ropiak et al., 2017).
Other sources of special CT types include:
• Leaves from several willow (Salix L. spp.) acces-sions and black currant (Ribes nigrum L.) have high proportions of PCs and PDs with trans-flavan-3-ol subunits, respectively (Porter, 1988).
• Tea [Camellia sinensis (L.) Kuntze] leaves and shea (Vitellaria paradoxa C. F. Gaertn.) nuts are unusual in
having high proportions of galloylated flavan-3-ol monomers and galloylated low molecular weight PDs (Henning et al., 2003; Ramsay et al., 2016).
• Water dock (Rumex hydrolapathum Huds.) roots and persimmon (Diospyros kaki Thunb.) fruits contain highly galloylated smaller PCs (mDP = 6; gal-loylation = 52%; Ropiak et al., 2016b) and larger PDs (mDP = 26; galloylation = 72%; Li et al., 2010), respectively.
• An entire series of oligomeric and polymeric PC xylosides are present in birch (Betula pendula Roth) bark (Liimatainen et al., 2012).
Not surprisingly, these biosynthetic patterns can generate contradictory and confounding effects when attempting to align CT structure with bioactivity (Laaksonen et al., 2015; Hixson et al., 2016). However, the particular CTs in these ‘specialist’ plants can provide unique opportunities for research, because it would be very difficult to separate sufficient quantities of a particular CT type from the complex CT mixtures, which are typical of most plants, for laboratory or in vitro studies. This problem is illustrated by the CT mixtures in different sainfoin, sericea lespedeza and Lotus accessions (Table 1), in which the PC/PD ratios ranged from 84:16 to 3:97, cis-/trans-flavan-3-ol ratios from 90:10 to 66:34, and mDP values from of 12 to 84 (Meagher et al., 2004; Stringano et al., 2012; Azuhnwi et al., 2013a; Mechineni et al., 2014).
An alternative approach is to use CTs from “CT specialist plants.” The already “pure” groups of either PCs or PDs, having either cis- or trans-flavan-3-ol stereochemistry, can be isolated from these specialist plants, separated in the laboratory into mDP variants, and used to explore the bioactivities of different PC/PD ratios, cis-/trans-flavan-3-ol ratios, and polymer sizes (Brown et al., 2017). Currently, this is the most straightforward approach to structure-activity studies, because chemical synthesis of CTs is even more challenging.
Another aspect of CT composition that is poorly researched concerns the galloylated CTs. This is a group of particularly potent antioxidants (Fig. 1; Li et al., 2010) that appear to possess strong antiparasitic, nematocidal, and antimicrobial activities but have received little attention for their nutritional or health effects (Brunet and Hoste 2006; Ropiak et al., 2016a). Acacia nilotica (L.) Delile leaves, carob (Ceratonia siliqua L.) pods, grape (Vitis vinifera L.) seeds, persimmon fruits, lentisk (Pistacia lentiscus L.) leaves, shea nuts, tea leaves, and Rumex L. spp. are good sources of galloylated CTs (Self et al., 1986; Henning et al., 2003; Papagiannopoulos et al., 2004; Spencer et al., 2007; Li et al., 2010; Rodríguez-Pérez et al., 2013; Derksen et
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It is also essential to use CT mixtures that are specific to the plant species being investigated, because CT composition affects ultraviolet-visible absorption maxima and reaction yields that result from the hydrochloric acid (HCl)-butanol-acetone and thiolysis assays (Grabber et al., 2013; Krueger et al., 2013; Engström et al., 2014; Hixson et al., 2015; Ropiak et al., 2016b; Wang et al., 2016). We emphasize the unsuitability of commercially available CTs from quebracho [Schinopsis quebracho-colorado (Schltdl.) F. A. Barkley & T. Mey.] as a standard because these have 5-deoxy-flavan-3-ol subunits, which give particularly low reaction yields that lead to overestimation of CT concentrations (Schofield et al., 2001; Rautio et al., 2007), and tannic acid is even less appropriate, as it contains none of the CT flavan-3-ol subunits.
Additional challenges include variation in extractability of CTs. Some can be extracted with water or aqueous methanol, and others require aqueous acetone, but many CTs are tightly bound to the plant matrix and cannot be extracted with these solvents. By using techniques that only measure the easily extractable CTs, researchers may risk missing a large fraction (Table 2) that
al., 2014; Ramsay et al., 2016; Ropiak et al., 2016b; our unpublished data).
PROGRESS IN TANNIN ANALYSISSeveral new techniques have been developed recently for analyzing CT mixtures. As Zeller (2019) has addressed this topic in detail, only a few additional techniques are described below. An important constraint in the quest for valid tannin data is the requirement for high purity standards for quantitation, which means that the CT concentration and purity of the standards needs to be assessed by CT-specific methods such as thiolysis (Gea et al., 2011; Grabber et al., 2013; Williams et al., 2014a) or nuclear magnetic resonance spectroscopy (Zeller et al., 2015a). We emphasize that the widely used elution of plant extracts with 70% aqueous acetone from Sephadex LH-20 columns for tannin “purification” can lead to CT concentrations as low as ~13 g CTs 100 g−1 “purified sample” (Williams et al., 2014b). Their use would overestimate CT concentrations, so additional steps are required to increase their purity (Stringano, 2011; Fryganas, 2016; Brown et al., 2017; Ropiak et al., 2017).
Table 1. Condensed tannin (CT) concentrations (g/100 g dry matter) and compositions of forage legumes and selected pasture plants (Note: these studies used various CT assays that were applied either to whole plants, isolated extracts, or purified CT fractions).
Plant species CT concentration PC/PD ratio† mDP‡ Referencesg 100 g−1 dry matter
Erect canary clover [Dorycnium rectum (L.) Ser.] 15–20 5:95–17:83 10–127 Sivakumaran et al., 2004
Sericea lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don]
6–13 3:97 33 Mechineni et al., 2014
Panicledleaf ticktrefoil [Desmodium paniculatum (L.) DC.]
22 nd§ nd Pawelek et al., 2008
Sulla (Hedysarum coronarium L.) 2–12 11:89–27:73 3–46 Tibe et al., 2011
Big trefoil (Lotus pedunculatus Cav.) 5–10 16:84–20:80 2–44 Meagher et al., 2004; Sivakumaran et al., 2006
Sainfoin (Onobrychis viciifolia Scop.) 1–9 5:95–50:50 12–84 Berard et al., 2011; Stringano et al., 2012; Azuhnwi et al., 2013a; Malisch et al., 2015
Prairie clover (Dalea purpurea Vent.) 4–9 nd nd Berard et al., 2011
Birdsfoot trefoil (Lotus corniculatus L. var. corniculatus)
0–5 60:40– 84:16 9 Meagher et al., 2004; Sivakumaran et al., 2006; Berard et al., 2011; Grabber et al., 2014
† PC/PD, procyanidin:prodelphinidin ratio.
‡ mDP, mean degree of polymerization.
§ nd, not determined.
Table 2. Overview of techniques for determining extractable or unextractable condensed tannins (CTs) and their composition.
Analytical technique†Extractable
CTsUnextractable
CTsInformation on CT
composition LiteratureHCl-butanol-acetone X X Limited Grabber et al., 2013Thiolysis, phloroglucinolysis X X X Guyot et al., 2001b; Gea et al., 2011; Hixson et al., 2015,
2016; Ramsay et al., 2016
NMR X X X Grabber et al., 2013; Zeller et al., 2015a
NIRS X X X Petersen et al., 1991; Larkin et al., 1997; Dykes et al., 2014; Grabber et al., 2014; Klongsiriwet 2016
MALDI-TOF MS X – X Stringano et al., 2011; Feliciano et al., 2012; Krueger et al., 2013
UPLC-MS/MS X – X Engström et al., 2014
† NMR, nuclear magnetic resonance; NIRS, near-infrared reflectance spectroscopy; MALDI TOF MS, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass spectrometry (for further information, see Zeller, 2019).
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may have potentially important bioactivities (Gea et al., 2011; Pérez-Jiménez and Lluís Torres 2011; Cheynier et al., 2015; Hixson et al., 2016; our unpublished data). The impact of extractable vs. unextractable CTs on ruminant nutrition and health has not yet been determined.
Thiolysis or phloroglucinolysis (analytical degradation of CTs with thiols or phloroglucinol) can be used to determine the composition of CTs by depolymerization, enabling characterization of the flavan-3-ol subunits (Zeller, 2019). The use of thiolysis to analyze CTs in whole plant material (removing the need for extraction) was first reported by Guyot et al. (2001b) for apple residues and then adapted to sainfoin and food samples (Hellström and Mattila, 2008; Gea et al., 2011). However, quantification remains problematic, and higher CT yields have been reported in some samples with the HCl-butanol-acetone assay than with the thiolysis or phloroglucinolysis assays. Low values have been linked to oxidative processes affecting reaction yields (Hixson et al., 2015; Klongsiriwet, 2016; Brillouet et al., 2017; Desrues et al., 2017).
However, thiolysis can also generate up to threefold higher yields than the HCl-butanol-acetone assay (Drake and Mueller-Harvey, unpublished data, 2016); these particular CTs had high degrees of galloylation (Fig. 1), and this demonstrates the variation in reactivity of CTs and yield of assayable end products. These inconsistencies illustrate the challenge of determining the amount and
type of CTs in forage material and reinforce the case for using more than one method for analyzing CTs, and for using plant-specific CT standards (see above). Recent observations also suggest that the optimum time for the in situ thiolysis is affected by the plant species. Some samples yielded more consistent CT parameters if thiolysis was performed for 2 to 3 h rather than 1 h, as suggested by Gea et al. (2011). Clearly, there is no one silver bullet for CT analysis, and methods need to be evaluated and adapted for a particular plant species and research objective.
Infrared spectroscopy techniques are of particular interest to plant breeders due to their speed and suitability for screening large numbers of samples; near-infrared reflectance spectroscopy (NIRS) can be used in the laboratory, whereas visible–near-infrared and shortwave-infrared spectroscopy have been used for field screening (Lehmann et al., 2015). Near-infrared reflectance spectroscopy has potential for measuring not only CT concentrations but also PC/PD and cis-/trans-flavan-3-ol ratios (Fig. 3) (Petersen et al., 1991; Larkin et al., 1997; Dykes et al., 2014; Grabber et al., 2014; Klongsiriwet, 2016; Mueller-Harvey et al., unpublished data, 2010). Sample analysis by NIRS is rapid but requires robust calibrations that are based on laboratory analyses. Once calibrated, a single NIRS scan can also generate a large amount of information on other nutritional parameters, such as fiber, protein, soluble carbohydrate,
Fig. 3. Near-infrared reflectance spectroscopy (NIRS) for predicting condensed tannin (CT) composition of sainfoin plants that had been analyzed by thiolysis (Gea et al., 2011; Mueller-Harvey et al., 2011).
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lignin, dry matter, and ash contents, as well as predicted digestibility and gross energy (Givens et al., 2000).
TANNIN VARIATION IN GERMPLASM COLLECTIONS AND POTENTIAL FOR TRAIT-DIRECTED PLANT BREEDINGAlignment of CT composition with function offers opportunities for exploiting their bioactivities, and germplasm collections offer a rich source of CT variation (Hayot Carbonero et al., 2011; Klongsiriwet, 2016). Concentrations of CTs vary greatly not only between plant species but also between accessions (Larkin et al., 1997; Mosjidis 2001; Sivakumaran et al., 2004; Gruber et al., 2008; Häring et al., 2008; Lorenz et al., 2010; Grabber et al., 2015; Hixson et al., 2016). Table 1 lists the variation in forage plants: birdsfoot trefoil tends to have the lowest (<5 g 100 g−1 dry matter) and sericea lespedeza and erect canary clover the highest CT concentrations (6– 20 g 100 g−1 dry matter).
The CT traits can also differ markedly between accessions, and between parts of the same plant (Springer et al., 2002). Examples are PC/PD ratios in sainfoin stem CTs of ~50:50 and in sainfoin leaf CTs of ~10:90 (Malisch et al., 2015). Although alfalfa (Medicago sativa L.) seed coats have CTs with mDP values of 4 to 7 and a PC/PD ratio of 93:7 (Koupai-Abyazani et al., 1993), only the smaller PC dimers and trimers have been detected in engineered alfalfa leaves (Hancock et al., 2012). This means that plant breeding can target CT composition and concentration, which is important because these traits have been linked to different bioactivities (see below).
Both concentration and composition can change with season (Theodoridou et al., 2011; Grabber et al., 2015; Muir et al., 2017), but accession differences tend to be much larger (Springer et al., 2002; Stringano et al., 2012). Importantly, environment did not affect the ranking of the CT traits of a few sainfoin accessions (i.e., there was no genotype ´ environment interaction) (Azuhnwi et al., 2013a ; Malisch et al., 2016); this demonstrates that there are opportunities for trait-directed breeding of new cultivars.
All enzymes involved in the biosynthesis of the CT building blocks (i.e., flavan-3-ols) have been identified, apart from the elusive final condensing enzyme(s) (Harding et al., 2013). Two genes and several myeloblastosis (MYB) transcription factors (i.e., proteins with myeloblastosis DNA-binding domains that regulate CT synthesis) are responsible for the production of two of the flavan-3-ols (i.e., catechin and epicatechin; Ferreyra et al., 2012; Cheynier et al., 2013; Zhu et al., 2015; Chezem and Clay, 2016), but the genes and transcription factors for the other flavan-3-ols await identification. The MYB transcription factors from barrelclover (Medicago truncatula Gaertn.) and rabbitfoot clover (Trifolium arvense L.) have been expressed in alfalfa and white
clover (Trifolium repens L.), leading to stable and heritable CT production in their shoots and leaves (Hancock et al., 2012, 2014; Verdier et al., 2012; Albert, 2015). These developments provide a pathway for introducing CTs into the leaves of alfalfa and white clover. It would be interesting to explore whether the CTs that are already expressed in white clover flowers can be expressed in the leaves.
It should also be possible to alter the CT composition through conventional crossing experiments. Scioneaux et al. (2011) showed that CT composition, especially the average polymer size (the mDP value), in Populus L. was controlled by genetics and that environment (location) and season (months) had only a small effect. In addition, interspecies hybridization can generate plants with novel CT traits, as demonstrated with narrowleaf trefoil (Lotus tenuis Waldst. & Kit. ex Willd.) ´ birdsfoot trefoil hybrids (Escaray et al., 2014).
Despite considerable advances in plant science, genomic resources for forage legumes are still scarce, especially for CT-containing forages (Szczyglowski and Stougaard, 2008; Hayot Carbonero, 2011; Zarrabian et al., 2013; De Vega et al., 2015; Mora-Ortiz, 2015). These are necessary to harness the potential benefits of CTs, and for breeding of new varieties with improved agronomic, nutritional, and antiparasitic traits. The European and Asian sainfoin germplasm is very diverse in terms of morphology, anatomy, drought resistance, CT traits, and genetic polymorphism (Hayot Carbonero, 2011; Zarrabian et al., 2013; Malisch et al., 2015, 2016; Mora-Ortiz, 2015; Kempf et al., 2016, 2017; Mora-Ortiz et al., 2016; Kölliker et al., 2017). For example, anatomical trait analysis (xylem/phloem ratio, vessel and sieve tube diameters, and phloem width) suggested that large differences exist in water and solute transport mechanisms between accessions, which are important for drought resistance (Zarrabian et al., 2013).
Molecular markers are helpful in the analysis of genetic diversity, mapping and quantitative trait loci analysis, and genomics-assisted breeding. Next-generation sequencing technology is facilitating the identification and use of molecular markers in plant genetics and breeding. RNA-sequencing technology is an efficient way of obtaining sequence information of all the genes that are expressed in a given plant tissue, and it can also be mined for molecular marker polymorphisms. The first such library of expressed genes in sainfoin was obtained from five accessions using this technology (Mora-Ortiz et al., 2016). Annotation of the expressed genes in the library allowed identification of 59 genes involved in the CT biosynthesis pathway (Mora-Ortiz et al., 2016). It also provided the platform for identifying >3800 simple sequence repeat (SSR) markers and 77,000 single-nucleotide polymorphism markers (Mora-Ortiz et al., 2016). Phylogenetic analysis revealed that sainfoin is closely related to red clover (Trifolium pratense L.) and barrelclover. Some of the SSR markers
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were used to assess the genetic diversity of European sainfoin accessions representing cultivars and noncultivars (ecotypes, landraces) (Kempf et al., 2016). These molecular markers are now available as tools for further genetic and genomic research. They were used to study inbreeding and self-fertilization in sainfoin. In a pilot study, one marker locus was identified that could explain up to 12% of the variation in CT composition (i.e., PC/PD ratio; Kempf et al., 2017). We anticipate that the sequence and molecular marker information now available (Kempf et al., 2016; Mora-Ortiz et al., 2016) can be used to assist breeding of novel sainfoin cultivars with CT traits that are optimized for animal nutrition and health.
AGRONOMY, WEED CONTROL, HARVESTING, AND PROCESSING OF TANNINIFEROUS FORAGE LEGUMESThere is currently considerable global interest in harnessing the benefits of CTs in forage legumes to support the sustainability agenda of agriculture; birdsfoot trefoil, sericea lespedeza, and prairie clover (Dalea purpurea Vent.) have been evaluated in the United States, Canada, New Zealand, and Switzerland (Mosjidis 2001; Waghorn 2008; Häring et al., 2008; Berard et al., 2011; Li et al., 2014; Grabber et al., 2015), sulla in Australia (de Koning et al., 2003, 2010; Heuzé et al., 2015) and sainfoin in Canada and Europe (Häring et al., 2008; Hayot Carbonero et al., 2011; Malisch et al., 2015; Bhattarai et al., 2016). The choice of which forage to grow will depend on the climate, soil, environment, and farming practices. The performance of these forages has been tested in pure stands and in combinations with partner species to increase the overall forage quantity and quality (Häring et al., 2008; de Koning et al., 2010; Hayot-Carbonero et al., 2011; Döring et al., 2013; Lüscher et al., 2014; Wang et al., 2015).
Cocultivation of Legumes and Companion CropsCocultivation of forage legumes with companion crops can deliver higher total yields (Nyfeler et al., 2009; Döring et al., 2013; Finn et al., 2013; Mora-Ortiz, 2015; Hunt et al., 2016; Malisch et al., 2017), more nitrogen fixed per hectare (Nyfeler et al., 2011; Vasileva and Ilieva, 2016) and improved dry matter and nitrogen digestibilities of coensiled mixtures (Wang et al., 2007), but success depends on the agronomic compatibility of the species, appropriate treatments, and development of new sainfoin cultivars with a more competitive canopy (Mora-Ortiz, 2015; Mora-Ortiz and Smith, 2016; Kölliker et al., 2017; Malisch et al., 2017).
Traditional mixtures for sainfoin establishment in the United Kingdom have included grasses such as timothy (Phleum L.) and meadow fescue [Schedonorus pratensis (Huds.)
P. Beauv.] or undersowing with spring barley (Hordeum L.) as companions (Liu et al., 2008; Mora-Ortiz, 2015; Mora-Ortiz and Smith, 2016). However, new investigations showed that chicory (Cichorium intybus L.)—which also has antiparasitic properties—and oat (Avena sativa L.) can be cocultivated with sainfoin for a short period (i.e., two agronomic cycles; Mora-Ortiz, 2015; Mora-Ortiz and Smith, 2016), but chicory was found to be a very aggressive partner and to suppress sainfoin (Häring et al., 2008). North American researchers have also explored oat and alfalfa as companion crops for irrigated birdsfoot trefoil and sainfoin (Wang et al., 2015; Hunt et al., 2016), and this involved developing a new sainfoin population for cocultivation with alfalfa. The novelty of this work lies in the fact that this new sainfoin population has good competitiveness against alfalfa compared with previous sainfoin accessions, where seedlings first produced a long taproot and could become outgrown by weeds and many companion species (Hayot Carbonero et al., 2011).
Weed ControlWeed control in sainfoin can be achieved (Mora-Ortiz, 2015; Mora-Ortiz and Smith, 2016; Malisch et al., 2017) through appropriate choice of partner species, sowing densities, and cutting frequencies and can lead to stable sainfoin percentages (i.e., ~40% of the sward was sainfoin), which suffice to reduce the incidence of bloat (Wang et al., 2006; Malisch et al., 2017). This was also demonstrated for several other legume species in a Pan-European experiment (Finn et al., 2013; Connolly et al., 2017; Suter et al., 2017). Other strategies for weed control can include application of preemergence, postemergence, and maintenance herbicides (Sheldrick and Thomson, 1982; Moyer et al., 1990; Frame et al., 1998; Amiri et al., 2013; Mora-Ortiz, 2015). In the case of sainfoin, weed suppression is directly correlated with sainfoin establishment and yields.
Current understanding of CT expression in sainfoin suggests that it should be possible to breed new forage legumes with good yields and consistent CT profiles, which is important, as farmers need varieties with predictable CT traits.
Effects of Drought on Yield and Condensed Tannin Content in Sainfoin AccessionsHowever, instead of using irrigation to boost yield, others have focused on exploiting the taproot of sainfoin, as sainfoin can remain productive on dry, marginal soils and can continue to grow during unseasonably dry weather. To test this drought tolerance, 30 sainfoin accessions were subjected to drought for 18 wk, with the mean soil water potential of the upper 40 cm being below −2 MPa, and were compared with the dry matter yields of rainfed controls that received additional irrigation when the soil water potential was below −0.6 MPa. The results were also compared with
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six other forage species (legumes, nonleguminous forbs, and grasses) of known drought tolerance. This showed that some sainfoin accessions were as tolerant under severe drought as alfalfa, and a few accessions even exceeded its drought tolerance. Most sainfoin accessions also had lower drought losses than chicory (Malisch et al., 2014). In addition, the CT traits were assessed under drought and control conditions for five of these accessions. Although CT composition was hardly affected by drought, the CT concentrations increased at the vegetative, but not the reproductive, stage (Malisch et al., 2016).
Moreover, there were no interactions between drought and accession for CT traits, and it remains to be seen whether the ranking of sainfoin accessions according to their CTs is maintained across other environments (Malisch et al., 2016). Another study with 100 sainfoin genotypes from 10 ecotypes showed that drought tolerance was, however, correlated with leaf proline content (Irani et al., 2015). This correlation is in accordance with previous studies, and although the cause-and-effect relationship between proline and drought tolerance is not yet fully understood, proline can act as an osmoprotectant, thus stabilizing membranes and maintaining cell turgor. Additionally, there is some indication that it might contribute to upregulation of drought tolerance genes (Szabados and Savouré, 2010; Per et al., 2017). Therefore, the current understanding of CT expression in sainfoin suggests that it should be possible to breed new cultivars with good yields, drought tolerance, and consistent CT profiles, which is important, as farmers need forage legumes with predictable CT traits.
Grazing vs. PreservationLegume swards expressing CTs can be grazed safely, as they are nonbloating (Wang et al., 2012; MacAdam and Villalba, 2015), but optimal use of CT forages would most likely be as a substitute for existing legumes in mixed swards, such as white clover with ryegrass (Lolium L.). This is because dry matter yields are greater from grasses than from legumes (subject to adequate nitrogen availability), and because farmers in temperate climates have identified forage species (including grasses) that enable profitable farming. In these situations, the CT concentration in the diet is diluted by the contribution of the CT-free component of the sward (Waghorn and Shelton, 1997), so high CT concentrations of the bioactive CT legume might be most useful, as the CTs in one forage can affect digestion of protein in the companion forage (Waghorn and Jones, 1989).
Several CT forages possess antiparasitic properties, which are of special interest for sheep and goat grazing systems (Hoste et al., 2010), or when there is anthelmintic resistance (Terrill et al., 2012). Condensed tannins may also provide benefits when immunity of animals is low (i.e., around parturition or weaning), but the need to combat parasitism at such times does not necessarily coincide with
the availability of fresh CT forages. Therefore, bioactivity needs to be maintained when processing CT forages into hay, pellets, or silages. Conserved forages allow out-of-season feeding, but may, especially in the case of pellets (Girard et al., 2016b), also offer opportunities for standardizing and optimizing CT traits and enable transportation to other regions.
Processing of CT forages into hay, pellets, or silages can have a marked effect on CT concentration and extractability (Fig. 4) and appears to increase protein-bound, and possibly covalently linked, CTs (Terrill et al., 1997; Minnée et al., 2002; Lorenz et al., 2010; Vernhet et al., 2011; Ramsay et al., 2015; Girard, 2016; Huang et al., 2016). Ensiling sainfoin or sulla reduced ammonia production, improved silage quality, and protected plant protein during fermentation, which improved its nutritional value relative to forages without CTs (Niezen et al., 1998b; Lorenz et al., 2010). There is a need for feeding trials to assess the biological significance of unextractable CTs in terms of ruminal or intestinal digestion and efficacy against parasitic nematodes. These questions could perhaps be addressed through experiments that explore accession differences in terms of unextractable CTs.
ELUCIDATING RELATIONSHIPS BETWEEN CONDENSED TANNIN STRUCTURES AND ANTIPARASITIC EFFECTSParasitism imposes a considerable nutritional penalty on animals, and therefore controlling the parasite burden will indirectly benefit the nutritional status of animals. This is the reason for noting that CT forages can be used for nutraceutical purposes, which refers to a combined action of nutritional and antiparasitic benefits that include anthelmintic (Terrill et al., 2012; Hoste et al., 2015, 2016) and anticoccidial effects (Kommuru et al., 2014; Saratsis et al., 2016). Most evidence of anthelmintic effects of CTs stems from in vitro assays, but some in vivo feeding trials
Fig. 4. Average percentages of extractable and unextractable condensed tannins (CTs) in fresh, pelleted, and ensiled sainfoin, sulla, and birdsfoot trefoil (Minnée et al., 2002; Lorenz et al., 2010; Ramsay et al., 2015).
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under controlled experimental conditions have also yielded promising results. It is therefore timely to summarize the emerging trends and CT structure-activity relationships, which were obtained with a range of in vitro assays, to guide future feeding trials.
Antiparasitic Effects of Prodelphinidins and Galloylated Condensed TanninsCondensed tannins with high molar percentages of PDs and galloylation have given good anthelmintic effects in vitro with parasite larvae (L). These include inhibition of L3 migration and L4 motility with Ascaris suum, as well as L1 feeding inhibition, adult motility assays of Ostertagia ostertagi and Cooperia oncophora, and larval exsheathment inhibition of Haemonchus contortus and Trichostrongylus colubriformis of L3 larvae (Brunet and Hoste 2006; Quijada et al., 2015; Desrues et al., 2016a; Ramsay et al., 2016). These findings could explain the excellent in vivo results with sheep or goats that were obtained when feeding sericea lespedeza (these CTs consist of almost pure PDs with high mDP values), big trefoil, sainfoin, sulla plants, or hazelnut (Corylus L.) peels (high PD percentage) and lentisk browse (galloylated compounds) (Landau et al., 2010; Rodríguez-Pérez et al., 2013; Hoste et al., 2015). These in vivo trials found better host resilience and lower fecal egg counts of H. contortus, Teladorsagia circumcincta, and T. colubriformis and of a mixed gastrointestinal nematode infection, which were attributed to lower worm fecundity (Niezen et al., 1995, 1998a; Paolini et al., 2005; Landau et al., 2010), and also lower Eimeria oocyst counts (Kommuru et al., 2014). Control of T. colubriformis was reflected in higher liveweight gains by lambs fed sulla than alfalfa (Niezen et al., 1995).
Antiparasitic Effects of the Mean Degree of Condensed Tannin PolymerizationThe average size of CTs in a polymeric mixture (mDP value) can also affect their antiparasitic activity, with larger polymers being more potent against exsheathment
of H. contortus L3 and larval feeding of O. ostertagi and C. oncophora L1 larvae in vitro (Quijada et al., 2015; Desrues et al., 2016a). However, PD-rich plants tend to have CTs with high mDP values and high CT concentrations (Kommuru et al., 2014; Laaksonen et al., 2015; Mueller-Harvey et al., unpublished data, 2016), confounding our understanding of CT structure-activity relationships. The characteristics of CTs responsible for antiparasitic effects need to be understood to identify optimal feed sources.
Apart from the examples above, very few other plants with high molar PD or galloyl percentages or high mDP values (>15) have been evaluated in vivo to determine their efficacy against gastrointestinal parasites. The plant kingdom remains a rich and underexplored resource of such promising CTs (Table 3), and it would be timely to test these in vitro results by feeding plants, browse, or agroindustrial byproducts with differing CT traits to parasitized livestock. As traits vary across accession and environment, it is important to analyze the dietary CTs to establish their relationships with antiparasitic effects. This would enable guidelines to be formulated for development of new plant cultivars for optimal bioactivities.
One other aspect also needs investigation: are CT traits that are optimal for antiparasitic effects compatible with nutritional benefits, or would farmers require plants with different CTs for either antiparasitic or nutritional purposes? Given the encouraging results with sericea lespedeza and panicledleaf ticktrefoil [Desmodium paniculatum (L.) DC.], especially in parasitized animals, anthelmintic and nutritional benefits may not be mutually exclusive (Terrill et al., 2012; Cherry et al., 2014), but other options could include sacrificing nutrition for a short period, enabling an appropriate period of CT feeding to achieve parasite control.
Mechanisms of Antiparasitic Condensed Tannin ActionA key question concerns the mechanisms by which CTs exert their effects. It is thought that the ability of CTs
Table 3. Plant materials containing condensed tannins (CTs) with structural characteristics that are of interest for conferring antiparasitic activities.
CT traits Plant species and part ReferenceProdelphinidin-rich CTs Peels of hazelnut (Corylus avellana L.) seeds; leaves of Persian ironwood
[Parrotia persica (DC.) C.A. Mey.], London plane (Platanus ´ hispanica Mill. ex Münchh. [occidentalis ´ orientalis]), black or red currant (Ribes nigrum L., R. rubrum L.) bushes, black locust (Robinia pseudoacacia L.); sainfoin
(Onobrychis viciifolia Scop.), sulla (Hedysarum coronarium L.), sericea lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don] plants; white clover
(Trifolium repens L.) flowers; erect canary clover [Dorycnium rectum (L.) Ser.]
Sivakumaran et al., 2004; Tibe et al., 2011; Mechineni et al., 2014; Hoste et al., 2016; Ropiak
et al., 2016b
Galloylated CTs Shea (Vitellaria paradoxa C. F. Gaertn.) nuts, persimmon (Diospyros kaki Thunb.) fruits, lentisk (Pistacia lentiscus L.) leaves, carob (Ceratonia siliqua L.) fruits, grape (Vitis vinifera L.) seeds, great water dock (Rumex
hydrolapathum Huds.) roots, dock (Rumex obtusifolius L.) leaves
Papagiannopoulos et al., 2004; Spencer et al., 2007; Li et al., 2010; Rodríguez-Pérez et al., 2013;
Ramsay et al., 2016; Ropiak et al., 2016b
High mean degree of polymerization values
Erect canary clover plant, persimmon fruits, apple (Malus domestica Borkh. spp., cider cultivars); leaves of Persian ironwood, London plane, black currant, black locust; white clover flowers; sericea lespedeza plant
Guyot et al., 2001a; Sivakumaran et al., 2004; Li et al., 2010; Mechineni et al., 2014; Hoste et al., 2016;
Ropiak et al., 2016b
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to suppress gastrointestinal nematodes stems from their ability to bind with proteins (Hoste et al., 2012). Thus, CTs could act by inhibiting key parasite enzymes, and indeed inhibition of parasite glutathione-S-transferases, which play an important role in the detoxification of xenobiotic compounds, has been confirmed in in vitro experiments (Hansen et al., 2016). Prodelphinidins have more phenolic groups capable of forming hydrogen bonds with proteins than PCs (Fig. 1), but it has also been established that the mDP value is the most important factor for CT protein aggregation and precipitation (Zeller et al., 2015b; Ropiak et al., 2017). Taken together, these observations could explain why PDs, which generally have larger mDP values than PCs, have better anthelmintic properties (Hoste et al., 2016; Kommuru et al., 2014, 2015).
By using electron microscopy, a number of studies have revealed physical deformations of H. contortus adult worms that had been exposed to CTs in either in vitro experiments or collected after feeding sericea lespedeza or sainfoin plants or tzalam [Lysiloma latisiliquum (L.) Benth.] leaves (Martínez-Ortíz-de-Montellano et al., 2013; Kommuru et al., 2015). The shriveled surfaces and plaque formations around orifices could account for inhibition of feeding and lowering of fecundity and are thought to stem from CTs interacting directly with proteins on parasite surfaces (Ropiak et al., 2016a). Scanning and transmission electron microscopy detected not only external but also internal damage to the cuticle and sensilla of the lip region of young and adult Caenorhabditis elegans by different CT types, and to the cuticle and underlying tissue (i.e., muscle cells) and intestinal cells of H. contortus L3 larvae and adults and of T. colubriformis L3 larvae by sainfoin and tzalam CTs (Brunet et al., 2011; Martínez-Ortíz-de-Montellano et al., 2013; Ropiak et al., 2016a). Given the low uptake (bioavailability) of polymeric CTs in mammalian tissues in comparison with monomeric flavonoids (Li and Hagerman, 2013), it would be worth exploring whether the internal tissue damage arises from the CTs or from other compounds that may be present in plant extracts (Brunet and Hoste, 2006; Williams et al., 2015; Klongsiriwet et al., 2015; Desrues et al., 2016a; Mengistu et al., 2017). It also raises the question whether mixtures of CTs plus co-occurring smaller plant compounds (such as quercetin, luteolin, cinnamaldehyde, etc.) can cause both external and internal damage, or whether internal damage may be due to a disruption of nematode metabolism, in turn causing necrosis of cells and tissues. Condensed tannins together with a flavone (luteolin), a flavonol (quercetin), or cinnamaldehyde can act synergistically against parasitic nematodes in vitro (Barrau et al., 2005; Klongsiriwet et al., 2015; Hoste et al., 2016; Ropiak et al., 2016a). These in vitro findings are yet to be tested in feeding trials using combinations of feeds with different CTs and such monomeric compounds, but such combinations should hopefully lead to future on-farm applications.
Tissue and Host Responses to Condensed TanninsIn addition to direct anthelmintic effects against parasites, more work is warranted on how CTs may influence host responses to parasites. Niezen et al. (2002) measured higher antibody titers against antigens to adult T. circumcincta and adult and larval T. colubriformis when lambs were fed with sulla compared with alfalfa. This may be a result of increased intestinal flow of proteins and amino acids associated with ruminal protection of protein degradation, which can contribute to the host’s ability to maintain growth, immune function, and regulation of worm populations (Ríos-De Álvarez et al., 2008; Ramírez-Restrepo et al., 2010; Hoste et al., 2012). Moreover, recent in vitro studies have also indicated that CTs can directly modulate the activity of immune cells such as gd T cells and dendritic cells, potentially enhancing the host’s innate immune response (Tibe et al., 2012; Williams et al., 2016, 2017). Similar to other bioactivity studies, immune-modulating activity in vitro is highly dependent on CT size, with mDP >6 eliciting a stronger response than CTs with mDP <6, and flavan-3-ol monomers have little or no effect (Williams et al., 2016, 2017).
Effects of Fermentation on Condensed Tannin ActivityWork has started on determining how fermentation affects CT concentrations and bioactivities. According to results from the HCl-butanol-acetone or thiolysis assays, fermentation can reduce “apparent” CT concentrations by 30 or 85%, respectively, in silages (Mena et al., 2015; Ramsay et al., 2015) and the gut (Desrues et al., 2017; Quijada et al., 2018). However, despite these apparent losses, sericea lespedeza and sainfoin silage extracts and silages still exerted anthelmintic effects in vitro by inhibiting the exsheathment of H. contortus L3 larvae and in vivo by lowering adult worm burden and fecal egg counts (Heckendorn et al., 2006; Manolaraki, 2011; Terrill et al., 2016). This may either be due to sufficient quantities of undegraded CTs or hydrolysis of anthelmintic flavonoids from inactive glycosides (Manolaraki, 2011), or to protein-bound CTs surviving ruminal fermentation and exerting anthelmintic activity in the abomasum, where a lower pH facilitates dissociation of the CT-protein complex ( Jones and Mangan, 1977). A high activity against O. ostertagi nematodes of the abomasum contrasted with no activity against intestinal Cooperia oncophora nematodes and was associated with 2.3% CTs (g 100 g−1 dry matter by thiolysis) in the abomasum vs. 0.02% CTs in the intestine (Desrues et al., 2016a, 2016b, 2017). However, both nematode species were affected by CTs in the in vitro larval feeding inhibition assay (Desrues et al., 2016a).
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EFFECTS ON RUMINAL FERMENTATION AND CONSEQUENCES FOR RUMINANT NUTRITION AND GREENHOUSE GAS EMISSIONSEffects on Nutrition and Dietary Protein UseNutritional effects of CTs are currently understood in general terms, and information concerning the ways that CTs affect specific processes are only now being defined by determining the CT traits of forages used for in vivo and in vitro measurements and their effects on the microbiome (Grosse Brinkhaus et al., 2016, 2017). Binding to dietary proteins and reduction in rumen proteolysis, resulting in higher proportions of dietary protein passing to the small intestine, have been measured (Waghorn, 2008), but details and mechanisms are not understood. The findings by Kariuki and Norton (2008) indicated that the release of dietary protein between the abomasum and terminal ileum was correlated with the protein precipitation capacity of CTs, which depends on the structures of both the CTs and the proteins (Hagerman and Butler, 1981; Dobreva et al., 2012). The measurements of Wang et al. (1996) are especially important in this regard, because they showed that in sheep fed birdsfoot trefoil with polyethylene glycol (PEG, which inactivated the CTs), ~80% of amino acid absorption occurred in the first half of the intestine, but the entire length of the intestine was required for absorption when CTs were active. Hence, the unknowns concerning CT interactions with rumen function, microbial growth, and intestinal absorption need to be evaluated in terms of dietary CT traits.
The current evidence suggests that only four forage legumes–birdsfoot trefoil, sainfoin, sulla and crownvetch [Securigera varia (L.) Lassen]–support higher ruminant growth rate or milk yield when fed as a sole diet, compared with CT-free diets (Burns et al., 1972; Waghorn et al., 1990, 1997; Mueller-Harvey, 2006; Rochfort et al., 2008; Waghorn, 2008; Patra and Saxena 2010; MacAdam et al., 2011; Naumann et al., 2013; Piluzza et al., 2014; MacAdam and Villalba 2015). However, other CT forage legumes or some CT browse species may be beneficial when fed as part of a diet (e.g., lotuses; Ayres et al., 2006). A universal consequence of dietary CTs is a reduction in urinary nitrogen excretion (because of reduced rumen proteolysis) and an increase in fecal excretion of nitrogen (Mueller-Harvey, 2006; Waghorn, 2008).
Evaluation of nutritional benefits can be complicated, especially if both feed quality (digestibility) and voluntary feed intake are important. Variations in intake are likely to confound comparisons of digestibility, but digestible matter intake is a recognized indicator of performance. There are a number of ways that the effects of CTs can be determined, and several studies (Table 4) have fed a CT forage to two groups of animals, with one receiving
daily doses (or intraruminal infusions) of PEG to bind and deactivate the CTs. This ensures that the majority of the diet is the same and enables the effect of the CTs to be evaluated. An option used by some researchers to compare a CT diet with a similar non-CT diet is fraught with difficulties. No species are optimal for comparisons, and any differences in composition, digestion, or intake will compromise the evaluation of the CTs. It is also important to distinguish the CT effect from a legume effect when evaluating a CT forage fed with grasses, for example, and if the animals are parasitized, any benefits of CTs could arise from direct or indirect impacts on the parasites (Hoste et al., 2015, 2016).
Furthermore, because CTs reduce the digestion of protein in the rumen and over the entire gastrointestinal tract (Waghorn, 2008), it is unlikely that any nutritional benefits will result when dietary crude protein is insufficient. Benefits are more likely when dietary protein is in excess of requirements. However, if in the presence of enough protein (or amino acids) other nutrients are limiting (e.g., energy intake or phosphorus), providing additional protein will not improve production (Waghorn, 2008; Pagán-Riestra et al., 2010). Hence, the methods by which animals are fed and their physiological state (e.g., lactating, growing, or at maintenance) when evaluating the nutritional effects of CTs can contribute to inconsistencies in findings. In addition, comparative measures of digestion may be confounded by variation in intakes, because increasing intakes may reduce digestibility (Tyrrell and Moe, 1975), but on other occasions have no effect (Hammond et al., 2013).
A number of studies with sainfoin and birdsfoot trefoil have also yielded contradictory results, often with lower or no production benefits measured when compared with CT-free controls (Thomson et al., 1971; Waghorn et al., 1997; Theodoridou et al., 2010; Aufrère et al., 2013; Azuhnwi et al., 2013b; Copani et al., 2016; Girard et al., 2016a, 2016b). In addition to the constraints mentioned above (dietary crude protein concentration, or whether other nutrients are limiting production), variations in CT traits within germplasms may also affect animal responses (Grabber et al., 2015). For example, the UK Hampshire Common and Cotswold Common sainfoin accessions had higher PC/PD ratios (>30:70) than the continental European Visnovsky accession (<19:81) (Stringano, 2011; Stringano et al., 2012) and could have accounted for these contradictory reports. Birdsfoot trefoil has delivered good growth rates in the United States and New Zealand, and the PC/PD ratios were ~80:20 (Meagher et al., 2004; MacAdam and Villalba, 2015). However, when two sainfoin cultivars with PC/PD ratios of 24:76 (Visnovsky) and 37:63 (Perly) were fed to lambs infected with H. contortus, concentrations of essential amino acids in plasma were higher than when both diets had
crop science, vol. 59, 2019 www.crops.org 13
been treated with PEG to inactivate CTs (Azuhnwi et al., 2013b); it is not known whether the higher plasma concentrations were a direct result of CTs on protein digestion or an anthelmintic effect against H. contortus. Variable results could be investigated by considering CT traits in conjunction with diet composition (e.g., protein, fiber, water-soluble carbohydrate, starch, and amino acid contents, as these can be affected by environment; Grabber et al., 2015), forage harvesting, or preservation methods. This means that nutritional evaluations of CT plants need to be undertaken under documented and controlled conditions, with information on harvesting and preservation methods (grazed, dried as hay or pellets, or ensiled).
Another potentially important issue could be how animals are fed, because CT traits also vary within plants. In Spain, it is traditional to take the first sainfoin cut as hay and then leave animals to graze the regrowth (Dr S.F. Demdoum, personal communication, 2010). Under light stocking regimes in the United Kingdom, sheep will only eat the sainfoin tops (flowers and younger leaves) and leave older leaves and stems intact (Mueller-Harvey, personal observation). Therefore, feeding whole plants as pellets, hay, or silage could result in forages with very different nutritive values than grazed forages, and also because conservation affects CT concentrations; these facts could be another reason for the contradictory results mentioned above.
One of the very few feeding trials that compared two birdsfoot trefoil cultivars with similar nutritional composition—apart from their CT concentrations—found that the ‘Maitland’ cultivar (3.5 g CTs 100 g−1 dry matter) achieved better nitrogen retention in sheep than the ‘Empire’ cultivar (0.5 g CTs 100 g−1 dry matter) (Waghorn et al., 1987). Greater retention of dietary protein suggested that the Maitland CTs protected more soluble protein from digestion in the
Tab
le 4
. Var
iatio
n o
f nut
ritio
nal e
ffec
ts a
chie
ved
with
diff
eren
t sai
nfo
in a
cces
sio
ns a
nd fe
edin
g re
gim
es in
co
mp
aris
on
with
two
tref
oil
spec
ies
and
cro
wnv
etch
fora
ge
leg
umes
.
Pla
nt s
pec
ies†
PC
/PD
‡m
DP
§N
utr
itio
nal
eff
ects
Ref
eren
ces
Sai
nfoi
n ac
cess
ions
: V
isno
vsky
(drie
d or
ens
iled)
19:8
1–24
:76
16–2
9La
mbs
: low
er a
ppar
ent o
rgan
ic m
atte
r and
fibe
r dig
estib
ility,
incr
ease
d pl
asm
a co
ncen
tratio
n of
ess
entia
l am
ino
acid
s, n
o ef
fect
on
body
N re
tent
ion
Sch
aren
berg
et a
l., 2
007;
Str
inga
no e
t al.,
201
2;
Azu
hnw
i et a
l., 2
013b
Per
ly (p
elle
ts, 2
0% o
f bas
al d
iet)
ND
ND
No
effe
ct o
n N
ret
entio
n in
dai
ry c
ows
Gro
sse
Brin
khau
s et
al.,
201
6
Per
ly (e
nsile
d w
ith ti
mot
hy g
rass
)N
DN
DLo
wer
gro
wth
rat
e of
lam
bs th
an o
n re
d cl
over
mix
ture
sC
opan
i et a
l., 2
016
Per
ly (e
nsile
d)N
DN
DC
onde
nsed
tann
ins
(CTs
) had
no
effe
ct o
n N
ret
entio
n co
mpa
red
with
the
+
poly
ethy
lene
gly
col (
PE
G) c
ontr
olTh
eodo
ridou
et a
l., 2
012
Sho
shon
e (g
raze
d)N
DN
DC
ompa
rabl
e w
eigh
t gai
ns b
y be
ef c
alve
s gr
azin
g sa
info
in a
nd a
lfalfa
die
tsM
augh
an e
t al.,
201
4; V
illal
ba e
t al.,
201
5
Ren
umex
(gra
zed)
ND
ND
Gro
wth
rate
and
sla
ught
er d
ata
wer
e th
e sa
me
for s
ainf
oin-
and
alfa
lfa-fe
d la
mbs
Kar
nezo
s et
al.,
199
4
No
deta
ils (f
resh
fora
ge)
ND
ND
CTs
in s
ulla
, but
not
in s
ainf
oin,
impr
oved
am
ino
acid
abs
orpt
ion
com
pare
d w
ith th
e +
PE
G c
ontr
olB
erm
ingh
am e
t al.,
200
1
Zeus
/Esp
arce
tte
(ens
iled)
ND
ND
Dai
ry c
ows:
hig
her
milk
yie
ld c
ompa
red
with
gra
ss s
ilage
Huy
en e
t al.,
201
6b
UK
acc
essi
ons
(Som
bour
ne,
Ham
pshi
re, C
otsw
old
Com
mon
)29
:71–
33:6
712
–32
Farm
ers
repo
rt h
igh
grow
th r
ates
of l
ambs
fed
UK
sai
nfoi
n ac
cess
ions
(but
no
acc
essi
on d
etai
ls w
ere
prov
ided
in fe
edin
g tr
ial)
Thom
pson
et a
l., 1
971;
Str
inga
no e
t al.,
201
2
Fak
ir-cu
t and
sta
ll-fe
dN
DN
DP
ositi
ve e
ffect
on
N re
tent
ion
com
pare
d w
ith r
ye g
rass
and
clo
ver,
grea
ter
recy
clin
g an
d de
grad
atio
n of
ure
a, im
prov
ed u
tiliz
atio
n of
recy
cled
NE
gan
and
Uly
att,
1980
Lotu
s sp
p.
Big
tref
oil (
Mak
u–in
door
tria
ls)
20:8
0N
DR
educ
ed g
row
th r
ate,
low
er a
ppar
ent d
iges
tibili
ty o
f ess
entia
l am
ino
acid
sW
agho
rn e
t al.,
199
7; M
in e
t al.,
200
3; M
eagh
er
et a
l., 2
004;
Wag
horn
, 200
8
Bird
sfoo
t tre
foil
(Gol
die
[indo
or tr
ials
], N
orce
n, O
berh
auns
tad
ter
[gra
zed
])84
:16
ND
Enh
ance
d ab
sorp
tion
of e
ssen
tial a
min
o ac
ids,
pos
itive
effe
cts
on li
vest
ock
prod
uctio
n (in
crea
sed
catt
le a
nd s
heep
gro
wth
, cow
and
she
ep m
ilk y
ield
, sh
eep
fert
ility
and
woo
l gro
wth
)
Wag
horn
and
She
lton,
199
7; W
agho
rn e
t al.,
199
7;
Mea
gher
et a
l., 2
004;
Wag
horn
, 200
8; M
acA
dam
et
al.,
201
1; M
acA
dam
and
Villa
lba,
201
5
Cro
wnv
etch
Che
mun
g, P
enng
ift (fi
eld
cure
d, w
ind-
row
ed, b
aled
)26
:74
>13
Larg
er d
aily
gai
n of
cat
tle a
nd s
heep
und
er g
razi
ng o
r st
all f
eedi
ng
com
pare
d w
ith a
lfalfa
or
seric
ea le
sped
eza.
Bur
ns e
t al.,
197
2, 1
977
(Not
e: o
ur C
T da
ta a
re
unpu
blis
hed
and
not f
rom
the
plan
ts th
at w
ere
fed)
† W
ith in
form
atio
n, if
ava
ilab
le, o
n va
riety
or
acce
ssio
n an
d m
etho
d of
feed
ing.
‡ P
C/P
D, p
rocy
anid
in:p
rod
elp
hini
din
rat
io; N
D, n
ot d
eter
min
ed.
§ m
DP,
mea
n d
egre
e of
pol
ymer
izat
ion.
14 www.crops.org crop science, vol. 59, 2019
rumen but did not interfere with protein digestion and absorption in the abomasum and small intestine. More research is needed to establish the precise fate of dietary protein in the abomasum and intestine in the presence of CTs. Estimates based on their potentially beneficial protein protection effects suggest that if alfalfa had just 1% CTs in its dry matter, this could achieve a 12% increase in net returns for US dairy farmers (McCaslin et al., 2014).
It is unfortunate that few studies describe the composition of CTs in plants that have been fed to ruminants, and the LegumePlus program has attempted to address this issue by encouraging communication and collaboration among chemists and plant and animal scientists. However, we still do not know which CT traits plant breeders should be targeting (i.e., dietary CT concentration, PC/PD ratio, or mDP values or a combination of these) to increase livestock production and product quality. Apart from a few in vitro studies (Azuhnwi et al., 2013b; Hatew et al., 2016; Huyen, 2016; Huyen et al., 2016b), no feeding trials have attempted to resolve the relationships between animal production and CT traits. Interdisciplinary research is needed to uncover the mechanisms by which CTs exert their effects on rumen (microbial) digestion and utilization of dietary nutrients, and this will require compositional (rather than colorimetric) analyses of the dietary CTs.
The size of CTs is the key parameter that controls CT protein aggregation and precipitation. This has been illustrated using bovine serum albumin (BSA) and gelatin, where aggregation increased markedly as the mDP values increased from 3 to 8 (corresponding to CT molecular weights of ~1000 to 2400 Da), but there were only small differences in the efficacies of larger CTs with mDP >9 to aggregate the proteins (Ropiak et al., 2017). It would be worth testing whether CT size also affects the mechanism by which CTs interact with dietary or endogenous animal proteins, as Zeller et al. (2015b) showed that relatively more alfalfa protein was precipitated by CTs than BSA protein, which could be due to the major leaf protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) being much larger (560 kDa) than BSA (67 kDa). It will be necessary to determine if CTs with different mDP values affect dietary protein degradation and digestion, especially as the major proteins in forages (RuBisCo), beans (albumins, prolamins), and intestinal gut tissue and mucoproteins have quite different structures, which will influence their interactions with CTs (Hagerman and Butler, 1981). As an aside, we have included bean proteins in this context, as several papers from the 1960s and 1970s on the nutritional effects of dietary tannins originated from feeding CT-containing beans ( Jansman, 1993; Lowry et al., 1996). It would therefore be timely to review some of these results in the light of new knowledge on CT and protein structures.
Effects on the Quality of Animal ProductsThere are some effects of sainfoin diets on milk and meat quality and on the animal’s physiological response to dietary CTs. Cattle fed a grass–sainfoin (mixture of Zeus and Esparcette accessions) silage or sheep grazed on big trefoil (‘Maku’) partitioned energy towards protein synthesis, rather than lipid synthesis, compared with grass–corn (Zea mays L.) silage and white clover diets, respectively (Purchas and Keogh, 1984; Huyen et al., 2016a). Feeding dairy cows with sainfoin pellets lowered milk and blood urea concentrations compared with alfalfa and birdsfoot trefoil pellets (Girard et al., 2016b; Grosse Brinkhaus et al., 2016) because of the reduction in rumen proteolysis and ammonia absorption.
Sainfoin and birdsfoot trefoil diets reduced bacterial biohydrogenation in the rumen, increased unsaturated fatty acid contents in milk, cheese, and meat products, and reduced indole and skatole in lamb meat (Priolo et al., 2005; Schreurs et al., 2007; MacAdam and Villalba, 2015; Girard et al., 2016a, 2016b; Huyen, 2016; Huyen et al., 2016a). Skatole and indole were associated with “fecal” flavor characteristic of pasture-fed products and originated from amino acid degradation by Clostridium aminophilum (Attwood et al., 2006), which was relatively sensitive to CTs (Sivakumaran et al., 2004). More recently, Campidonico et al. (2016) reported that CTs and polyphenol oxidases in a sainfoin–red clover silage mixture generated additive effects that increased the intramuscular unsaturated fatty acid contents of lambs compared with a pure grass diet.
Effects on Nitrogen and Methane EmissionsThe reduction in urinary nitrogen and increase in fecal nitrogen excretion seem to be universal consequences of dietary CTs fed to ruminants and are important because a greater proportion of nitrogen is lost from urine than feces. Thus, CTs can improve soil nitrogen status, lower emissions of the potent greenhouse gas nitrous oxide (N2O), and lessen nitrogen leachate into waterways and groundwater (Kingston-Smith et al., 2010; Theodoridou et al., 2010). A shift from urinary to fecal nitrogen could redue nitrogen losses by 25% and achieve savings on nitrogen fertilizers based on preliminary estimates from the Integrated Farm System model for dairy farms (Zeller and Grabber, 2015). Other opportunities for reducing the environmental impact of nitrogen emissions from ruminant livestock include the use of galloylated CTs and epigallocatechin gallate (a galloylated flavan-3-ol monomer), because they are urease inhibitors (Huynh-Ba et al., 1994; Powell et al., 2011; Takeuchi et al., 2014). Urease inhibition reduces ammonia emissions from urine and subsequent N2O production (Kingston-Smith et al., 2010). Grape seeds and some agroindustrial residues are sources of galloylated CTs and epigallocatechin gallate (Li et al., 2010; Lee et al., 2014; Ramsay et al., 2016), and
crop science, vol. 59, 2019 www.crops.org 15
application to the barn floor could lessen ammonia and N2O emissions from intensive systems.
It is clear that CTs can provide important benefits to ruminant farming; however, high dietary concentrations or CTs with the “wrong” compositional traits will lower the digestion and utilization of dietary protein and absorption of essential amino acids by ruminants (Min et al., 2003). The challenge is to identify which CT traits are best able to enhance utilization of dietary protein to improve animal production, environmental sustainability, and profitability for farmers.
CONCLUSIONS AND POSSIBLE DIRECTIONS FOR THE FUTURECondensed tannins are the fourth largest group of secondary plant metabolites in the plant kingdom and provide opportunities for breeding forage legumes with novel CT traits. Research on CT-containing feeds has the potential to improve ruminant health by preventing bloat and mitigating effects of parasitism, as well as lowering environmental footprints and improving the sustainability of food quality and production for consumers (Tedeschi et al., 2014). Innovative molecular approaches have enabled alfalfa and white clover to express PC dimers and trimers in their foliages (Hancock et al., 2012, 2014; Verdier et al., 2012; McCaslin et al., 2014). Apart from research by Mosjidis and colleagues, who selected for low CT concentration and grazing-tolerant sericea lespedeza for cattle production (Mosjidis, 2001), there has been hardly any plant breeding for enhanced CT composition; however, progress is becoming feasible, as new genomic data and molecular markers for CTs have been obtained (Kempf et al., 2016; Mora-Ortiz et al., 2016). Alternatively, cultivars with specific CT traits could also be obtained by conventional selection, focusing on the compositional CT differences that already exist between plant species and between, but also within, accessions and plant parts.
This review has highlighted that interdisciplinary research is essential for developing new forages with desirable CT traits and bioactivities, and that it requires well-coordinated inputs from plant scientists, chemists, animal nutritionists, and parasitologists. Such collaborations have succeeded in identifying the large variation in CT traits and their in vitro nutritional and parasitological effects that pertain to the germplasms of sainfoin and birdsfoot trefoil (Stringano et al., 2012; Grabber et al., 2014; Malisch et al., 2015), which grow in temperate climates. Such variation is likely to exist also in other species (e.g., sericea lespedeza and prairie clover, which grow in warm humid and colder regions, respectively; Mosjidis, 2001; Berard et al., 2011). It would be timely to explore these in vitro results by feeding CT forage legumes with varying CT traits to develop robust targets and tools for plant breeding.
Researchers must become aware that most colorimetric assays are not appropriate for determining CT concentrations or compositions. For example the p-dimethylaminocinnamaldehyde and HCl-vanillin reagents detect CTs, but also their monomeric flavan-3-ol precursors, and the Folin–Ciocalteu reagent detects all phenolic groups, whether in monomeric flavonoids, proteins, or condensed or hydrolyzable tannins (Schofield et al., 2001). These problems are made worse by the use of inappropriate CT material to construct calibration curves for analysis of CT concentrations, as mentioned above (Grabber et al., 2013; Krueger et al., 2013). The fact that a multitude of methods or standards have been chosen to measure CTs has prevented comparisons of published CT concentrations from feeding trials and the setting of optimum thresholds for CT traits. Appropriate CT analysis is crucial to progressing this field.
Laboratory studies have probed the impact of CT concentration, polymer size, and PC/PD ratios on ruminal fermentation and antiparasitic effects. The time has come for feeding trials with selected forages of similar nutritional compositions but different CT traits to test these in vitro results and establish the in vivo nutritional and antiparasitic effects associated with contrasting CT traits. This will also require comparison of forages that have been grazed or processed into hay, pellets, or silages, because CTs become less extractable on processing, but the underlying mechanisms and biological significance of these changes are yet to be explored. Such studies will help to optimize dietary protein utilization and energy partitioning and reduce the environmental footprint of livestock production.
We also need answers to the following questions: what effects do CTs exert on intestinal cells in ruminants and nonruminants in terms of nutrient absorption and cell signaling cascades? What are the mechanisms by which CTs protect dietary protein from rumen degradation and affect amino acid absorption from the intestine (e.g., in the presence of CTs, amino acid absorption takes place across the entire intestine; however, in the absence of CTs, absorption occurs in the first third of the intestine; Wang et al., 1996)? What are the effects on the ruminal or colonic microbiomes? How does the impact of CTs on recycling of urea-nitrogen modify the utilization of energy and protein in dairy cows, and what is the origin of higher fecal nitrogen outputs in the presence of CTs? We need to establish the fate and bioactivity of CTs during ensiling and digestion, their mechanisms of action against parasites along the digestive tract, and their effects on the in vivo immune response and establish relationships with CT concentration and composition. This review has also highlighted the need for analytical methods and CT standards that are fit for purpose so that published CT values can be compared between research groups and experiments.
16 www.crops.org crop science, vol. 59, 2019
Although considerable progress has been achieved over recent years, questions remain on how to translate the research results into practice, such as:
• How should we use CT forages? Is it better to graze forages with low CT concentrations and use high-CT forages as supplements?
• Can high-CT and CT-free forages be grazed together in strips, and could this improve utiliza-tion of dietary protein?
• Can the competitiveness and persistency of CT-containing legumes be increased so they can be sown together with other crops and achieve suc-cessful weed suppression?
• What are the ecological implications of intake and selectivity by different ruminant species on plant persistence in the field?
• Does an optimum CT concentration and com-position exist that can deliver nutritional plus antiparasitic effects? According to Cherry et al. (2014), it may be possible to achieve both, or do we need forages with different CT compositions (i.e., cultivars that either improve protein utiliza-tion or possess antiparasitic effects)?
• Will increased use of CT forages generate resistance of parasites to CTs, and how can feeding regimes mitigate against this?
• Is it best to use a short-term supply of high-CT for-ages for reducing parasite burdens at strategic times (e.g., pregnancy, parturition, weaning), or should a longer-term supply of low-CT forages be used to boost the immune response?
• What agronomic, harvesting, or processing mea-sures can best ensure that CT plants deliver consistent results? Which cultivars should be grazed and processed into hay, pellets, or silage, and at what times of the year?
From these questions we need to develop practical solutions through collaboration with farmers and veterinarians. Producers and consumers alike are looking for sustainable innovations that produce high-quality foods profitably while also maintaining soil fertility and the quality of our environment.
Conflict of InterestThe authors declare that there is no conflict of interest.
AcknowledgmentsThis work was supported by a European Union Marie Curie training network (“LegumePlus”, PITN-GA-2011-289377), the Danish Council for Independent Research (Technology and Production Sciences, Grant no. 12–126630), the Institut National de la Recherche Agronomique, France (Drastic and Sustainable Treatment Reduction against Parasitism in Livestock project from the ‘Gestion Intégrée de la Santé Animale’ Metaprogramme), and in part by a USDA-ARS Specific Cooperative Agreement no. 58-3655-0-155F with the University of Reading, UK. We are grateful to the USDA-AR GRIN germplasm database for providing seeds of the sainfoin accessions.
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